JP5208513B2 - Implantable biomaterial and method of producing the same - Google Patents

Description

The present invention relates to an implantable biomaterial and a method for producing the same. In particular, the present invention can be used on or in conjunction with an implantable device, and can be used in a method of making the same, which reduces and / or alleviates biomaterial calcification. And a method for treating a collagen-containing biomaterial for improving the long life.

There are many methods for chemically altering and / or immobilizing the collagen matrix of biological tissues so that these tissues can be transplanted into a living mammalian body. There is. Examples of altered or fixed implantable biological tissues include heart valves, blood vessels, pericardium, skin, dura mater, tendons, and ligaments.

These biological tissues mainly consist of collagen and elastin. The stiffness / elasticity of most biological tissues is largely determined by the relative content of collagen and elastin contained in the tissues and / or the physical arrangement of the connective tissue framework.

Each collagen molecule is composed of three polypeptide chains that are intertwined to form a coiled triple helix. Biological tissue preservation chemicals generally form crosslinks not only between adjacent collagen molecules (intermolecular), but also between amino groups in polypeptide chains within collagen molecules (intramolecular).

Collagen-based biomaterials are prone to hyperacute rejection when used as a device that can be implanted in different recipient species. This hyperacute rejection is a natural immune reaction, triggered by an antigen present in the structure of this collagen-based biomaterial. Hyperacute rejection is a rapid degenerative process that affects the function and durability of such implantable devices.

The antigenicity of collagen-based biomaterials can be suppressed by physical or chemical crosslinking of the collagen. Physical crosslinking methods such as UV irradiation and heat dehydration result in low density crosslinking. For this reason, chemical agents such as formaldehyde, glutaraldehyde, oxidized starch, and certain polyepoxy compounds have been used as chemical cross-linking agents for collagen-based biomaterials.

Collagen cross-linking results from the reaction of cross-linking agents at the amine groups of lysine or hydroxy lysine residues on different polypeptide chains. Another known collagen cross-linking method is a method in which a carboxyl group of a glutamic acid residue and an aspartic acid residue is activated with a polypeptide chain and reacted with an amino group of another polypeptide chain to form an amide bond. .

Crosslinking can also be accomplished by crosslinking the amine groups of adjacent polypeptide chains with diisocyanates. This method is less common due to the toxicity and constant solubility of most diisocyanates.

In recent years, glutaraldehyde has been chosen as a cross-linking agent. Glutaraldehyde is bifunctional due to the presence of aldehydes present at both ends of the 5 carbon aliphatic chain. Apart from fixing tissue, glutaraldehyde is also an excellent sterilant for preparing biological tissue for transplantation.

In particular, permanent implantable biomaterials fixed with glutaraldehyde include porcine living heart valves, bovine pericardial valves, and bovine pericardial patches.

A problem associated with implanting biological materials that are cross-linked with chemicals is that these materials, particularly those with collagen and elastin in the material, tend to calcify. The calcification of these materials results in hardening and, when hardened, results in decomposition and breakage of the material. Both extrinsic and intrinsic calcification are responsible for the calcification of the crosslinked biomaterial.

Unfortunately, glutaraldehyde is known to promote calcification of biomaterials. The reaction of aldehydes and primary amines in the biomaterial forms unstable imines (Schiff bases), which subsequently release glutaraldehyde from the biomaterial. Unbound aldehydes present in the tissue can cause severe tissue stimulating effects such as an inflammatory response after transplantation. As such, it is necessary to remove or inactivate the calcification promoting effect of a crosslinking agent such as glutaraldehyde.

The mechanism of calcification of cross-linked biomaterials has not yet been fully understood. Clinical data show that factors such as patient age, infection, host tissue chemistry, dehydration, strain, dietary factors, and inappropriate initial anticoagulant therapy promote calcification of transplanted biomaterials. Show you get.

Many attempts have been made to find ways to mitigate the calcification of crosslinked biomaterials. Research on mitigation of calcification of biomaterials has mainly focused on the treatment of cross-linked biomaterials and is described in, but not limited to, the following patents and literature. US Patent No. 4,553,974 (Dewanji et al.), US Patent No. 4,120,649 (Schechter), US Patent No. 4,648,881 (Naschev et al.), And U.S. Pat. No. 4,976,733 (Girard), Vuyavare et al., 95, pages 479-488, published in 1997, and Circulation J. Biomed. Mater Res. It is described in Pasak and other papers published in Volume 69A, pages 140-144. These publications generally describe how to treat fixed tissue with alcohol prior to transplantation. In other words, the tissue is already cross-linked before exposure to alcohol. Even in cases where the tissue is pre-cultured in the presence of alcohol, the exposure time is often too short to be useful, otherwise buffers or other agents can adversely affect cross-linking stability. (See, for example, the 1997 paper by Vyavare et al. Above). Other processes have also been published for immobilizing biomaterials with non-glutaraldehyde reagents, including but not all of the following. That is, polyglycidal ethers (Paper 17: 1101 to 1103 by Imamura et al. Published in Jp. J. Artif. Organs, published in 1988), photooxidants (J. Biomed. Mater., Published in 1994). Res., Moore et al., 28, pages 611-618).

Treating cross-linked biomaterials with amino diphosphates and surfactants has been demonstrated to reduce calcification of these biomaterials after transplantation. However, these agents tend to be washed away from the biomaterial after transplantation and merely delay calcification.

The use of alcohol for biomaterial treatment is well known, but its use is limited to use as a solvent and / or sterilant. For example, the use of alcohol in the treatment of biomaterials to combat pathological calcification is limited to the use in advance of cross-linked collagen biomaterials, such as US Pat. No. 5,746,775 (Levy, et al. ) And International Patent No. WO84 / 01894.

Under such circumstances, there is still a need for a method for producing a biomaterial having resistance to calcification for a long time.
U.S. Pat. No. 4,553,974 US Patent No. 4,120,649 U.S. Pat. No. 4,648,881 US Patent No. 5,746,775 International Patent No. WO84 / 01894 Vyavare et al., Circulation, 1997, 95, 479-488. Pasack et al. Biomed. Mater Res. 2007, 69A: 140-144. Imamura et al., Jpn. J. et al. Artif. Organs, 1988, 17: 1101-1103 Moore et al. Biomed. Mater. Res. 1994, 28: 611-618.

The inventors have developed a method that overcomes or at least reduces the calcification problem of implantable biomaterials containing collagen.

And in the first aspect, the present invention provides:
A method of making an implantable biomaterial comprising:
(A) providing a method comprising exposing the biomaterial to an alcohol-containing solution for at least 24 hours;

In a second aspect, the present invention provides:
A method of treating a biomaterial comprising collagen to produce an anti-calcified biomaterial comprising the steps of:
(A) providing a method comprising exposing the biomaterial to an alcohol-containing solution for at least 24 hours;

In some embodiments, the method of the first and second aspects further comprises
(B) exposing the biomaterial to a cross-linking agent in step (a);
(C) exposing the biomaterial to an acidic solution in step (b);
Steps (b) and (c) are sequentially performed after step (a).

On the other hand, step (a) is preferably performed for at least 24 hours, more preferably for at least 36 hours, and most preferably for at least 48 hours, and under certain circumstances, step (a) is shorter. One skilled in the art will appreciate that it may be performed only in time.

And in a third aspect, the present invention provides:
A method of making an implantable biomaterial comprising:
(A) exposing the biomaterial to an alcohol-containing solution;
(B) exposing the biomaterial to a cross-linking agent in step (a);
(C) exposing the biomaterial to an acidic solution in step (b);
Steps (b) and (c) provide a method of sequentially performing after step (a).

In a fourth aspect, the present invention provides:
A method for treating a collagen-containing biomaterial to produce an anti-calcified biomaterial comprising:
(A) exposing the biomaterial to an alcohol-containing solution;
(B) exposing the biomaterial to a cross-linking agent in step (a);
(C) exposing the biomaterial to an acidic solution in step (b);
Steps (b) and (c) provide a method that is sequentially performed after step (a).

In some embodiments, the biomaterial or collagen-containing biomaterial between step (a) and step (b) and / or between step (b) and step (c) Is washed away to remove residual alcohol and / or residual crosslinker. Desirably, the biomaterial or collagen-containing biomaterial is further washed away after step (c) to remove residual acidic solution.

One skilled in the art will appreciate that the methods disclosed herein are useful for the treatment of any biomaterial. Desirably, the biomaterial comprises collagen.

In some embodiments, the biomaterial is a cultured tissue, or a prosthesis comprising an extracellular matrix obtained from an animal, or a regenerated tissue (eg, collagen matrix), or the like.

It will be appreciated that the biomaterial may have synthetic analogs formed from synthetic polymers, including those commonly found in natural tissue matrices, or biopolymers, or both. Suitable polymers include, for example, polyamides and polysulfones. The biopolymer can be naturally generated or produced in a test tube, for example, by fermentation.

In some embodiments, the biomaterial is naturally occurring and has been removed from the animal. The biomaterial can be extracted from any animal, whether from the same species as the recipient or from an animal of a different species than the recipient. Desirably, the animal is one of the order of mammals, ie, mammal eyes, artiodactylae, rabbit eyes, rodents, odd-hoofed eyes, carnivores and marsupials. More desirably, the animal is selected from the group consisting of sheep, cows, goats, horses, pigs, marsupials and humans.

The biomaterial may be any type of cellular tissue. Desirably, this cellular tissue is cardiovascular tissue, heart tissue, heart valve, aortic root, aortic wall, aortic apex, pericardial tissue, connective tissue, dura mater, skin tissue, vascular tissue, cartilage, pericardium, ligament, Selected from tendons, blood vessels, umbilical tissue, bone tissue, fascia, and submucosa and skin.

In some embodiments, the biomaterial is and / or has discrete or separated collagen rather than spontaneous collagen-containing tissue. Discrete collagen can be used in a separated state, or can form a medical device or medical article as is known in the art.

The biomaterial used in step (a) has not been cross-linked so far.

The alcohol-containing solution used in step (a) is desirably a liquid and water-based solution that is an aqueous solution having a volume percentage of alcohol greater than about 50%, more preferably 60% alcohol. To 80%. Either a buffered alcohol-containing solution or a non-buffered alcohol-containing solution can be used. However, since it has been shown that buffered alcohol-containing solutions have the adverse effect of producing a yellowed biomaterial in the subsequent crosslinking step, it is desirable to use unbuffered alcohol-containing solutions.

In the method of the present invention, any alcohol known in the technical field of alcohol-containing solutions can be used. Desirably, the alcohol is a C ₁ -C ₆ lower alcohol in a buffer-free solution. More desirably, the alcohol is selected from the group consisting of methanol, ethanol, cyclohexanol, isopropanol, propanol, butanol, pentanol, isobutanol, 2-butanol and tert-butanol.

In some embodiments, the alcohol-containing solution has a mixture of two or more alcohols, the total volume of the mixture being greater than 50%. For example, a mixture of about 70% ethanol and about 10% isobutanol is effective.

The biomaterial in step (a) can be exposed to the alcohol-containing solution for a time long enough to allow the biomaterial to be resistant to in vivo pathogenic calcification. Desirably, the biomaterial remains in contact with the alcohol-containing solution for a time sufficient for alcohol to diffuse and penetrate into the biomaterial. More desirably, the biomaterial is exposed to the alcohol-containing solution for at least 24 hours, even more desirably for at least 36 hours, and most desirably for at least 48 hours.

For example, in embodiments where the biomaterial is also exposed to the alcohol-containing solution for longer than 24 hours, the biomaterial may be used as such in the treatment method of the invention disclosed below. Good.

In some embodiments, the biomaterial is exposed to one or more cross-linking agents after removal of the alcohol-containing solution and the solution is removed. Any type of cross-linking agent or combination thereof known in the art is used for a time sufficient to cross-link the collagen. It will be understood that the crosslinking agents include, but are not limited to: That is, divinyl sulfone (DVS), polyethylene glycol divinyl sulfone (VS-PEG-VS), hydroxyethyl divinyl sulfone methacrylate (HEMA-DIS-HEMA), formaldehyde, glutaraldehyde, aldehydes, isocyanates, alkyl halides Aryl halides, imide esters, N-substituted maleimides, acylated compounds, carbodiimides, chlorides, N-hydroxysuccinimides, light (eg, blue light and ultraviolet light), pH, temperature, and combinations thereof. Desirably, the cross-linking agent is a chemical cross-linking agent selected from the group consisting of carbodiimide, polyepoxy ether, divinyl sulfone (DVS), polyaldehyde, and diphenylfophoryl azide (DPPA).

In some embodiments, the polyaldehyde is bialdehyde, or trialdehyde, or dialdehyde. Glutaraldehyde is particularly preferred.

In some embodiments, the crosslinking step (b) is followed by step (c), but may or may not involve a flushing step. The acidic solution used in step (c) inactivates both or one or both of the crosslinker components present in the biomaterial after step (b). And / or any modification that contains any acid that removes or reduces the calcium binding sites obtained. Alternatively, or in addition, the acidic solution used in step (c) contains some acid that can further crosslink the active carboxyl group to the active amino group on the collagen to form an amide bond. . Desirably, the acid in the acidic solution is an aminocarboxylic acid. Desirably, the aminocarboxylic acid has at least one amino group and at least one carboxylic acid substituent. More preferably, the aminocarboxylic acid is selected from the group consisting of L-arginine, L-lysine, L-histidine, L-glutamic acid, or L-aspartic acid.

In some embodiments, step (c) of the disclosed method is substituted or supplemented with a method that inhibits the formation of metalloproteinases on the elastin molecules present in the biomaterial. In particular, a higher proportion of elastin is present in tissues such as aortic tissue than in other tissues. These elastin molecules can provide sites for the formation of metalloproteinases, and these sites need to be reduced, removed or inactivated. As shown in Example 1 below, buffer-free solutions containing polyvalent cations such as ferric salts and aluminum salts can be used to reduce metalloproteinase formation.

The step of washing away the biomaterial is performed using phosphorus-free 0.9% saline.

In one desirable embodiment, the biomaterial after step (c) is further sterilized. More desirably, the biomaterial is sterilized after flushing.

It will be appreciated by those skilled in the art that the temperature at which the steps of the present invention are performed is not critical, but desirably the temperature is between 2 ° C. and 40 ° C. It will also be appreciated that more desirably between 4 ° C and 30 ° C, most desirably between 5 ° C and 25 ° C.

In one embodiment, the alcohol, crosslinker and acidic solution, rinse solution, and sterile solution are all buffer free.

One skilled in the art will appreciate that the methods disclosed herein can produce anticalcified biomaterials that retain less than about 50 μg of calcium per mg of tissue for longer than 200 days after in vivo implantation. . In other words, the anti-calcified biomaterial of the present invention can be transplanted for longer than 200 days without the biomaterial increasing the tissue calcium content to greater than 50 μg / mg.

Accordingly, in a fifth aspect, the present invention provides an anti-calcified biomaterial having cross-linked collagen, comprising less than about 50 μg of calcium per mg of the biomaterial, and further comprising a calcium content per mg of the biomaterial. It provides a biomaterial that can be transplanted for at least 200 days without increasing to greater than 50 μg.

If it is not desired to be bound by any theory or assumption, as disclosed herein, the observed anti-calcified biomaterial is partly due to the method of making the biomaterial. This is caused by the presence of a synthetic polymer in the prepared collagen.

Accordingly, in a sixth aspect, the present invention provides an implantable biodevice having an anticalcified biomaterial having cross-linked collagen, wherein the collagen comprises a synthetic polymer.

In some embodiments, the anti-calcified biomaterial is coated on the surface of a medical device. In yet another embodiment, the device has at least a second coating.

One skilled in the art will appreciate that at least the second coating has an agent such as an antibacterial or antiviral or antifungal or growth factor or antidehydrating or disinfecting agent.

Desirably, the antibacterial agent is isoniazid, ethambutol, pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones, ofloxacin, sparfloxacin, rifampin, azithromycin, clarithromycin, dapsone, tetracycline, erythromycin, ciprofloxacin, Doxycycline, ampicillin, amphotericin B, ketoconazole, fluconazole, pyrimethamine, sulfadiazine, clindamycin, lincomycin, pentamidine, atovaquone, paromomycin, diclazaryl, acyclovir, trifluorouridine, foscarnet, penicillin, gentamicin, ganciclovir, iatroconazole Miconazole, zinc pyrithione, gold, platinum, silver and zinc Heavy metals including, but not limited to, and salts such as chlorides, bromides, iodides and periodates, and other complex forms, including supported complexes, and other forms Selected from the group consisting of

Desirably, the growth factor drug is hydroxyapatite, basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), nerve growth factor (NGF), epidermal growth factor (EGF), insulin-like growth. Factors 1 and 2 (IGF-1 and IGF-2), platelet derived growth factor (PDGF), tumor angiogenic factor (TAF), vascular endothelial growth factor (VEGF), corticotropin releasing factor (CRF), transforming growth factor α And β (TGF-α and TGF-β) selected from the group consisting of interleukin-8 (IL-8) or granulocyte macrophage colony stimulating factor (GM-CSF) or interleukins, and interferons .

In some embodiments, the device of the present invention further comprises a bioabsorbable material, the bioabsorbable material comprising polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polydioxanone, poly Caprolactone, polypeptides,, polycarbonates, polyhydroxybutyrate, poly (alkylene oxalate), copolymers of vinyl acetate with unsaturated carboxylic acids, water-soluble or water-dispersible cellulose derivatives, ethylene oxide polymers , Polyacrylamide, collagen, gelatin, poly (orthoester), amino acid polyamides, polyvinyl alcohol, polyvinyl pyrrolidone, polyetheretherketone, tricalcium phosphate, and mixtures thereof.

It will be appreciated that the device of the present invention may be any device where anticalcification is desired. Desirably, the device is an artificial heart, an extracardiac heart assist device, a self-contained or external heart assist device, a heart valve prosthesis, an annuloplasty ring, a skin graft, a vascular graft, a vascular stent, a structural stent, a vascular shunt , Cardiovascular shunt, dural graft, cartilage graft, cartilage graft, pericardial piece, ligament prosthesis, tendon prosthesis, bladder prosthesis, cotton ball, suture, permanent indwelling percutaneous device, surgical patch, cardiovascular One selected from the group consisting of a stent, a covered stent, and a covered catheter.

In some embodiments, the device further comprises a piece of tissue taken from an animal or a synthetic similar tissue.

Desirably, the tissue piece includes a plurality of cells, and upon implantation at the surgical site, the plurality of cells proliferate and integrate with surrounding tissue.

In a seventh aspect, the present invention provides a biocompatible implant, the biocompatible implant has a biocompatible scaffold with an anti-calcified biomaterial having cross-linked collagen; The biomaterial has a calcium content of less than about 50 μg / mg biomaterial, and the biomaterial does not increase its calcium content to greater than about 50 μg / mg biomaterial and is transplanted for at least 200 days Is possible.

In an eighth aspect, the present invention provides a biocompatible implant, the biocompatible implant has a biocompatible scaffold with an anti-calcified biomaterial having cross-linked collagen; The collagen has a synthetic polymer.

Desirably, the implant of the present invention further comprises a synthetic polymer, natural polymer, injectable gel, ceramic material, self-generated tissue, allogeneic tissue, xenogeneic tissue, and combinations thereof.

In a ninth aspect, the present invention provides a tissue injury repair kit, which comprises:
(A) one or more anti-calcification biomaterials having cross-linked collagen, wherein the collagen contains a biomaterial having a synthetic polymer;
(B) a description of the injury subject;
Have

In a tenth aspect, the present invention provides a method for treating living tissue, the method of treatment comprising:
(A) providing an anti-calcified biomaterial having cross-linked collagen, wherein the collagen has a synthetic polymer;
(B) transplanting the biomaterial into a patient in need of treatment;
including.

The tissue treatment method can be any treatment, including prophylactic treatment and therapeutic treatment.
Desirably, the tissue treatment method is selected from the group consisting of tissue repair, deep tissue protection, tissue swelling, cosmetic surgery, therapeutic treatment, tissue augmentation, and tissue sealing.

In an eleventh aspect, the present invention provides a wound dressing, which is a wound dressing having an anti-calcified biomaterial having cross-linked collagen, the collagen having a synthetic polymer. Is.

The biomaterial collagen is selected from the group consisting of sheep collagen, bovine collagen, goat collagen, equine collagen, porcine collagen, marsupial collagen and human collagen.

In some embodiments, the wound dressing further comprises a sulfated polysaccharide, the sulfated polysaccharide being heparin, chondroitin sulfate, dextran sulfate, dermatan sulfate, heparan sulfate, keratan sulfate, hexuroni It is selected from the group consisting of ruhexosaminoglycan sulfate, inositol hexasulfate, and sucrose octasulfate. Desirably, the wound dressing further comprises an antibacterial agent, antiviral agent, growth factor, anti-dehydrating agent, or disinfectant.

Desirably, the biomaterial has at least 50% collagen, more desirably 70% collagen, and most desirably consists essentially of collagen.

Before describing the present invention in detail, it is to be understood that the present invention is not limited to the illustrated method of preparation, and can of course vary. Further, the terms used in the present specification are only used to describe special embodiments of the present invention, and are further limited to those limited only by the appended claims. It should also be understood that it is not intended to be added.

All publications, patents, and patent applications cited above or below in this application are incorporated by reference in their entirety. However, the publications mentioned herein are cited to describe and disclose procedures and reagents that are reported in such publications and that may be used in connection with the present invention. Is. Nothing in this application should be construed as an admission that the invention is not entitled to antedate its disclosure by virtue of prior invention.

In addition, the practice of the present invention uses conventional immunization techniques, chemistry, and pharmacology within the art, unless otherwise specified. Such techniques are known to those skilled in the art and are fully explained in the literature. These include, for example, Corian, Dan, Ploe, Spicer and Wingfield, “Modern Procedures of Protein Science” 1990, Volumes I and II (John Willy and Sons); E. Bailey and D.C. F. Auris "Basics of Biochemical Technology" McGraw-Hill Book Company, New York, 1986, "Immunochemical Methods in Cellular and Molecular Biology" (Edited by Meyer and Walker, Academic Press, London, 1987), "Experimental Immunology Handbook, Volumes I-IV (DM Weir and CC Blackwell, 1986).

It should be noted that as used in this specification and the appended claims, the use of the singular includes the plural references unless the context clearly dictates otherwise. For example, a reference to “crosslinking agent” includes a plurality of such agents, a reference to “alcohol” refers to a reference to one or more alcohols, and so on. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice and test the present invention, the preferred materials and methods are now described. .

In one of the broadest aspects, the present invention relates to a method of making an implantable biomaterial.

As used herein, the term “biomaterial” refers to any material that has bioavailability, where the material comprises some type of collagen. This collagen may be any type of collagen derived from any one, and may be present alone or in combination with other materials. And, this collagen may have a weight ratio of only 1% to the total weight of the biomaterial, or may be 100%.

The term “collagen” is used herein to refer to an extracellular family of fibrous proteins characterized by a rigid triple helical structure. Three collagen polypeptide chains (“α chains”) wrap around each other to form this helical molecule. The term is also used to encompass various types of collagen.

The major part of the collagen helix varies little between mammals. In fact, many collagen types have a high degree of nucleotide and amino acid sequence homology. For example, the nucleotide sequence homology of collagen alpha I type II is at least 88% when compared to that of humans, horses and murines. Between humans and horses, there is 93% sequence homology at the nucleotide level, while mice and horses have 93% sequence homology. Nucleotide sequence homology between humans and mice is 88% (National Center for Biotechnology Information accession numbers U62528 (horse), NM033150 (human), and NM031163 (murine), http: //www.ncbi.nlm.nih.nih. gov). Other types of collagen have similar levels of amino acid homology. For example, the nucleotide sequence homology between porcine collagen alpha I type I and sheep collagen alpha I type I is 90% ((National Biotechnology Information Center accession numbers AF29287 (sheep) and AF201723 (pig), http://www.ncbi.nlm.nih.gov).

Given the common ancestry of many of the above-mentioned animals and the biological level of high amino acid and nucleotide sequence homology for collagen across many species such as cattle, sheep, rabbits and pigs, one skilled in the art It will be appreciated that the method of making a biomaterial as disclosed herein is applicable to collagen material isolated from all mammals.

Thus, in some embodiments, the biomaterial is isolated or harvested from a single mammal eye, ie, cloven-hoofed, rabbit-eyed, rodent, odd-hoofed, carnivorous, and marsupial It has been done. The animal is desirably a sheep, cow, goat, horse, pig, marsupial or human. While the biomaterial is desirably isolated from the same animal species as the recipient, the biomaterial is also expected to be isolated from an animal different from the recipient.

In some other embodiments, the biomaterial has cultured tissue or reconstituted tissue or the like.

The biomaterial may be any type of cellular tissue. For example, this cellular tissue can be cardiovascular tissue, pelvic floor tissue, heart tissue, heart valve, aortic root, aortic wall, aortic apex, pericardial tissue, connective tissue, soft or solid organ matrix, dura mater, skin tissue, Vascular tissue, dura mater, cartilage, pericardium, ligament, tendon, blood vessel, umbilical tissue, bone tissue, fascia, and submucosa or skin.

It will be appreciated that the biomaterial can include synthetic analogs formed from synthetic polymers, purified biopolymers, or both, including those commonly found in natural tissue matrices. . Suitable synthetic polymers include, for example, polyamides and polysulfones. The biopolymer can be naturally generated or produced in a test tube, for example, by fermentation.

The purified biopolymer can be suitably formed in the substrate by techniques such as weaving, knitting, casting, molding, extrusion, cell alignment, and magnetic alignment. Suitable biopolymers include, but are not limited to, collagen, elastin, silk amino acid, keratin, gelatin, polyamino acids, polysaccharides (eg, cellulose and starch), and copolymers of any of these. Not a thing. For example, the collagen polymer and the elastin polymer can form a synthetic implantable material using any of the techniques described above, such as weaving and molding. Synthetic tissue analogs mimic the natural tissue matrix. Alternatively, synthetic substrates can be used alone or with naturally occurring substrates to form tissue analogs, such as polypropylene, polylactic acid, nylon, silicon and the like. However, it is not limited to these.

Although the methods disclosed herein have some effect on pre-crosslinked biomaterials, the methods disclosed herein are not crosslinked, ie, “natural It is intended to be used for "unprocessed" or "unprocessed" materials.

Once the biomaterial is available, it is ready for transplantation. The terms “transplant”, “transplantable”, and “transplant” are used interchangeably herein, all of which are those of the biomaterial, device, etc. It refers to the ability to be placed in or on living tissue without introducing rejection, infection or toxicity problems. It should be understood that the term “implantable” can include a portion of a biomaterial or device, and can also include a partially implanted device, such as a contact lens.

In the first step of the method of the invention, the biomaterial is exposed to an alcohol-containing solution. As used herein, the term “exposed” or “exposed” refers to a biomaterial or collagen-containing material as described herein or in an alcohol-containing solution as described below. Refers to the aggressive step of contacting, followed by contacting the biomaterial with a crosslinking agent or acidic solution or other material for a time sufficient to produce the desired result. Methods for exposing biomaterials to, for example, alcohol-containing solutions are known to those skilled in the art. For example, in general, biomaterials can be “exposed” to alcohol by spraying or by immersion in a solution with alcohol.

As used herein, the term “alcohol” refers to any alcohol known to those skilled in the art, which alcohol removes or reduces the amount of triglyceroids and the carboxyls found on collagen. The group is partially esterified. Desirably, the alcohol is a water soluble alcohol. More preferably, the alcohol is a C ₁ -C ₆ lower alcohols buffer-free solution. Even more desirably, the alcohol is selected from the group consisting of methanol, ethanol, cyclohexanol, isopropanol, propanol, butanol, pentanol, isobutanol, 2-butanol and tert-butanol.

Without wishing to be bound by any particular theory or hypothesis, we believe that alcohol-containing solutions help unravel the triple helix of collagen, thereby helping to expose hydrophobic sites. (See Karita and Nishida, Biochim Biophys Acta., 1979, 23; 581 (1), pages 106-113). The inventors further believe that the carboxyl and amine groups found in collagen are esterified in the presence of an alcohol-containing solution to allow crosslinking in a later step. As such, the desired alcohol solution is at least about 50% v / v by volume, more preferably at least about 70% v / v by volume, and most preferably at least volume, relative to the buffer free solution. It has about 80% v / v alcohol in proportion. In one embodiment, the alcohol solution is 70% v / v ethanol by volume in 0.9% saline (containing 0.5 mM phenylmethylsulfonyl fluoride).

In certain embodiments, the methods of the present invention provide a method of making an implantable biomaterial that includes at least 24 in an alcohol-containing buffer-free solution having less than 100% alcohol. Exposure over time.

In some embodiments, it is postulated that the aldehyde-containing cross-linking agent reacts with the buffer during immobilization to cause the tenth of this aldehyde, so like other solutions and reagents, Alcohol-containing solutions are “buffer free”.

The step of exposing the biomaterial to an alcohol-containing solution allows the biomaterial to be resistant to in vivo pathogenic calcification, and the majority (ie high proportion) of carboxyl and amine groups found in collagen. As long as it is long enough to be esterified, it can be run for any time. Desirably, the biomaterial remains in contact with the alcohol-containing solution for a time sufficient for the alcohol to diffuse and penetrate into the biomaterial. More desirably, the biomaterial is exposed to the alcohol-containing solution for at least 24 hours, even more desirably at least 36 hours, and most desirably at least 48 hours.

Once this biomaterial is exposed to alcohol, the alcohol is removed. In some embodiments, the biomaterial is washed away with a wash solution having 0.9% saline without phosphorus after alcohol exposure. Nevertheless, any physiologically acceptable solution that is not buffered may be used as the washing solution. This is not so critical because the purpose of the cleaning solution is to remove excess alcohol.

This biomaterial or collagen-containing material can be used for transplantation immediately after being exposed to alcohol for longer than 24 hours. While alcohol pre-immobilization has been used, it has traditionally been used to sterilize tissue rather than esterify the carboxyl and amine groups found on collagen. As such, until now, the time of exposure to amine groups has been relatively short, i.e. less than 24 hours, which is insufficient for the alcohol to fully penetrate the tissue. As a result, prior to the present invention, an anti-calcification biomaterial (see below for definition) can be made by exposing the biomaterial to an alcohol-containing solution for a longer period of time, ie, longer than 24 hours. There was no correct recognition. This means that the biomaterial can be implanted as an anti-calcified biomaterial (described below) immediately after exposure to an alcohol-containing solution for longer than 24 hours. However, those skilled in the art will appreciate that an excellent form of anti-calcified biomaterial can be made by cross-linking of the biomaterial after step (a).

Thus, in some embodiments of the invention, the biomaterial or collagen-containing material after exposure to alcohol is then exposed to one or more bifunctional crosslinkers. As used herein, “bifunctional” refers to two functional aldehyde groups present at both ends of a five carbon chain. Crosslinking can be performed using any type of crosslinking agent that can crosslink collagen by any technique known in the art. The crosslinking agents include, but are not limited to: That is, acylated compounds, chlorides, aldehydes, alkyl halides and aryl halides, bisimidates, carbodiimides, divinyl sulfone (DVS), formaldehyde, glutaraldehyde, glyoxal, hexamethylene diisocyanic acid, hydroxide chloride, Hydroxyethyldivinylsulfone methacrylate (HEMA-DIS-HEMA), isocyanates, imide esters, light (eg, blue light and ultraviolet light), N-hydroxysuccinimide, N-substituted maleimides, pH, polyaldehyde, diphenylphosphori Ruazide (DPPA), polyepoxy compounds having important elements of 17-25 carbon and 4-5 epoxy groups, polyepoxy ethers, polyethylene glycol divinyl sulfone (VS-PEG-VS) Polyglycerol polyglycidyl ethers, temperature and combinations thereof.

In some embodiments, the cross-linking agent is a chemical cross-linking agent such as carbodiimide, polyepoxy ether, divinyl sulfone (DVS), genipin, glutaraldehyde, formaldehyde, and diphenylfophoryl azide (DPPA).

It has been demonstrated to date that polyepoxy compounds having a backbone of 17-25 carbon and 4-5 epoxy groups exhibit high performance in cross-linking collagen (eg, US Patent Application No. 20040059430 (S / N 10/618, 447)). It shows that the toxicity of polyepoxy compounds is lower than that of glutaraldehyde, and that antigenic or immune response induction decreases in proportion to reaction time when reacting to helical peptide molecules such as collagen. It has been. Of course, this polyepoxy compound exhibits relatively good biocompatibility (for example, Lore et al., 1992, Artif. Organs, 16: 630-633 and Uematsu et al., 1998, Artif.Organs, 22 pp. 909-913). In conclusion, the above-described polyepoxy compound is one of the desirable cross-linking agents.

In some embodiments, the cross-linking agent has about 1% glutaraldehyde and the length of exposure time is at least 24 hours. It will be appreciated that the length of time that the biomaterial is exposed to the cross-linking agent depends on the drug used, the concentration, and the temperature. Typically, the exposure time is between 24 hours and 28 days. The exact amount of exposure of the biomaterial to the cross-linking agent can be determined to the extent that one skilled in the art can fully accommodate.

Again, unless it is desired to be constrained by any particular theory or hypothesis, the inventors can expose biomaterials that have been exposed to alcohol to a cross-linking agent on the collagen present in the biomaterial. It is believed that the esterified carboxyl group and the amine group are crosslinked.

It will be appreciated by those skilled in the art that the temperature at which the steps of the present invention are performed is not critical, but preferably the temperature is between 2 ° C and 40 ° C. It will also be appreciated that more desirably between 4 ° C. and 30 ° C., most desirably between 5 ° C. and 25 ° C.

After crosslinking, once again the biomaterial is desirably washed away with a cleaning solution as used after the alcohol exposure step (a). However, it will also be appreciated that the flush step is merely more desirable.

Following the cross-linking step or, if a wash-out step is utilized after cross-linking, the biomaterial is exposed to an acidic solution containing any acid to remove or reduce the resulting calcium binding sites. However, this acid inactivates and modifies both the immobilized and non-immobilized crosslinker components present in the biomaterial after step (b). Either or both can be done. In addition to this, or in addition to this, the acidic solution used in step (c) may further comprise any acid that can crosslink the active carboxyl group to the active amine group of collagen to form an amide bond. Including.

Desirably, the acidic solution has at least one aminocarboxylic acid. As used herein, the term “aminocarboxylic acid” is any acid having at least one amino group and at least one carboxylic acid substituent. Representative examples of aminocarboxylic acids useful in the present invention include, but are not limited to, L-glutamic acid, L-aspartic acid, L-lysine, L-arginine, and L-histidine. There are two factors in the use of acidic solutions. The first is to assist in the inactivation and / or modification of fixed and non-fixed crosslinkers. The second element is to crosslink the active carboxyl group to the active amine group of collagen to form an amide bond.

The concentration of aminocarboxylic acid depends on the actual acid used and other parameters such as the total mass of biomaterial used. Furthermore, the wet weight ratio of aminocarboxylic acid to biomaterial is about 1: 4. The most important aspect of the acidic solution is pH. The pH must be lower than pH 7, desirably lower than pH 6, more desirably lower than pH 5, and most desirably lower than about pH 4.6.

In one embodiment, the acidic solution is 8 mg aminocarboxylic acid per milliliter of deionized water, the acidic solution is phosphorus free and has a pH of about 4.

The biomaterial is exposed to the aminocarboxylic acid for a period of at least 6 hours, more desirably at least 24 hours, and even more desirably 48 hours. The culture temperature is not critical, but is preferably between 5 ° C and 55 ° C, more preferably between 10 ° C and 45 ° C, and most preferably about 45 ° C.

In some embodiments, step (c) of the disclosed method is replaced or supplemented by a method that inhibits the formation of metalloproteinases on elastin molecules present in the biomaterial. In particular, a higher proportion of elastin is present in tissues such as aortic tissue than in other tissues. These elastin molecules can provide sites for metalloproteinase formation, such that these sites must be reduced or removed or inactivated. As shown in Example 1 below, buffer-free solutions containing polyvalent cations such as magnesium, ferric salts, and aluminum salts can be used to reduce metalloproteinase formation.

After exposing the biomaterial or collagen-containing material to an acidic solution and / or a buffer-free solution containing a polyvalent cation, the biomaterial is desirably washed away with another wash solution. In some embodiments, the biomaterial is further sterilized.

The biomaterial sterilization step is by any sterilization method known in the art for collagen-containing materials. For example, the biomaterial may be exposed to a sterilant (eg, a liquid sterilant such as a 0.2-2.0% weight ratio glutaraldehyde solution). A 0.625% glutaraldehyde solution can also be used as a sterilant in combination with heat (ie, at a temperature above room temperature but below the temperature that damages the biomaterial). Alternatively, a suitable sterilant solution is one that has a combination of only an osmotic equilibrium aqueous solution or non-contact sterilization reduction (eg, for radiation, electron beam, ultraviolet light, or other similar purposes). Alternatively, it may contain an aqueous glutaraldehyde solution combined with phosphate buffered saline. In examples where a 0.625% glutaraldehyde solution is used as the sterilant, the sterilization period may be 1 to 6 days at 37 ° C, or 1 to 2 days at 50 ° C. This final sterilization step may be performed after the biomaterial has been packed into its final container, so that there is a need for some processing of the biomaterial that is subsequently performed by the time of implantation. Disappear.

In certain desirable embodiments, the biomaterial is sterilized by exposing the biomaterial to 0.25% glutaraldehyde in deionized water containing 9.07 g / l potassium dihydrogen phosphate buffer.

The sterilization step may be performed for any amount of time and may include storage. The sterilization temperature is desirably carried out between 40-50 ° C. for longer than 60 minutes.

The biomaterial, after being treated with the methods disclosed herein, has a high resistance to calcification, ie, “anti-calcified biomaterial”. As used herein, the term “calcification” refers to one of the major pathological problems associated with biomaterials with connective tissue proteins (ie, collagen and elastin) made by conventional methods. Point to. It has been shown so far that these materials may calcify after implantation in the body. Such calcification can result in undesirable curing or degradation of the biomaterial. The exact mechanism by which such calcification occurs is unknown, but it is known that there are two types of calcification: intrinsic and extrinsic calcification in fixed collagen biomaterials . Endogenous calcification is characterized by the precipitation of calcium ions and phosphate ions in a fixed biological prosthetic tissue containing collagen matrix and remnant cells. Exogenous calcification involves the growth of cells (eg, platelets) attached to the biomaterial and calcium phosphate-containing surface plaques on the biomaterial, where calcium ions and phosphate ions precipitate within the attached thrombus. There is a feature to do.

For this reason, when the phrase “high resistance to calcification” or “anticalcification” is used in the biomaterial of the present invention, the biomaterial is at least 200 days after transplanted in vivo. This means that the amount of calcium per mg of tissue dried after removal is less than 50 μg, desirably less than 20 μg, more desirably less than 10 μg.

Desirably, the biomaterial of the present invention is resistant to enzymatic degradation. As used herein, the term “resistance to enzymatic degradation” refers to the ability of the biomaterial of the present invention to withstand enzymatic degradation to a level comparable to tissue fixed by conventional methods.

Once the biomaterial is formed, the implantable biomaterial or collagen-containing material of the present invention can be used to treat a number of diseases and / or disorders.

Here, the terms “treat”, “treatment” and the like are used in the present specification to act on an individual or an animal, and the tissue or cell obtains both pharmacological and / or physiological effects. Refers to that. The effect is particularly therapeutic in terms of partial or complete healing of both the disease and the disease, or either. As used herein, “treating” is directed to any treatment of vertebrates, mammals, particularly human diseases and / or disorders, and includes any of the following. (A) To suppress the disease and / or the disease, that is, to prevent its progression. Or (b) removing or ameliorating the symptoms of both the disease and the disease, or either, i.e., enzymatic degradation / both the disease and the disease, or the causative degeneration of either of the symptoms.

“Disease” and / or “Disease” are used herein interchangeably and refer to an abnormal condition that affects animals, including humans, using the biomaterials of the present invention. Can be treated. Accordingly, treatment of trauma, lesions, tissue degeneration, microbial infection, burns, ulcers, skin diseases are included in the present invention. Furthermore, the heart valve, aortic root, aortic wall, aortic apex, pericardial tissue, connective tissue, dura mater, skin tissue, vascular tissue, cartilage, pericardium, ligament, tendon, blood vessel, umbilical tissue, bone tissue, fascia and Submucosal replacement is included.

The anti-calcified biomaterial of the present invention can also be applied to any of a wide variety of contacting surfaces of a medical device. This contacting surface includes, but is not limited to, a surface intended to contact blood, cells or other body fluids or tissues of animals including humans. Suitable contact surfaces include one or more surfaces of a medical device intended to contact blood or other tissue. This medical device includes aneurysm coils, artificial blood vessels, artificial hearts, artificial valves, artificial valves, artificial kidneys, artificial tendons and artificial ligaments, blood bags, blood enzyme addition devices, bone and cardiovascular substitutes, bone prostheses, bone wax, heart Vascular grafts, cartilage replacement devices, catheters, contact lenses, cells and tissue culture and regeneration containers, embolic particles, filter systems, grafts, guide channels, indwelling catheters, laboratory instruments, microbeads, nerve growth guides , Ophthalmic implants, orthopedic implants, pacemaker leads, probes, prostheses, shunts, stents, peptide supports, surgical instruments, sutures, syringes, urinary tract substitutes, wound dressings, wound dressings , Wound healing devices, and other medical devices known in the art.

Other examples of medical devices that would benefit from applying the present invention will be apparent to those skilled in the art of surgery and medical procedures and are also contemplated by the present invention. The contact surface can be a mesh, coil, wire, inflatable balloon, or any other structure that includes targets within a vessel, a lumen, a solid tissue, etc. It can be transplanted to a site. These devices are delivered by or incorporated into intravascular catheters and other medical catheters.

The surface coating treatment of such a device can be carried out by a plasma coating method, as described in International Patent Application No. WO 96/24392.

In one best embodiment, the biomaterial of the present invention is used directly as a wound dressing. For example, as described above, the biomaterial can be used directly as a wound dressing after drying.

The wound dressing of the present invention is preferably in the form of a continuous sheet, similar to wound dressings known in the art. However, the present invention may take other specific forms. For example, the wound dressing of the present invention may be produced by cutting out a desired design pattern from the biomaterial inventory sheet described above. Further, for example, the mold may be cut from a biomaterial stock sheet.

In use, the wound dressing of the present invention is desirably used as a primary dressing which is in direct contact with the wound bed or set as close as possible to the wound bed. This dressing may also serve as a packaging material and, if necessary, is fixed in position with any suitable secondary wound dressing or with a device such as a wrap, tape, gauze, pad, suture or clip. May be. This dressing may be temporary or permanent and may be permanently incorporated into the healing tissue. If necessary, the wound dressing modifies the wound dressing by first removing the dressing top material and then removing the dressing to remove accumulated necrotic tissue and exudate. The temporary wound dressing of the present invention can be replaced with an unused dressing or other suitable wound dressing.

This dressing can also be placed directly in the wound. The dressing of the present invention can be cut, shaped, and modified to suit many uses and applications.

A further use of the biomaterial of the present invention is in the delivery of therapeutic benefit agents included in any of the applications described above. This therapeutic agent can participate in and improve the wound healing process and may include antibacterial agents, including antifungal, antibacterial, antiviral and antiviral agents. Parasite drugs, growth factors, angiogenic factors, anti-inflammatory drugs, antithrombotic drugs, anesthetics, mucopolysaccharides, metals, and other wound healing drugs include but are not limited to these.

Examples of antibacterial agents that can be used in the present invention include isoniazid, ethambutol, pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones, ofloxacin, sparfloxacin, rifampicin, azithromycin, clarithromycin, dapthorn, tetracycline, erythromycin , Ciprofloxacin, doxycycline, ampicillin, amphotericin B, ketoconazole, fluconazole, pyrimethamine, sulfadiazine, clindamycin, lincomycin, pentamidine, atovaquone, paromomycin, diclazaryl, acyclovir, trifluorouridine, foscalnet, penicillin, gentamicin, Ganciclovir, iatroconazole, miconazole, zinc piriti Heavy metals including, but not limited to, gold, platinum, silver, zinc, and copper, and composites of the above, including salts such as chloride, bromide, iodide, periodate, and supported complexes There are forms, and other forms, but are not limited thereto.

Growth factor agents incorporated into the wound dressing device of the present invention include basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), nerve growth factor (NGF), epidermal growth factor (EGF), Insulin-like growth factors 1 and 2 (IGF-1 and IGF-2), platelet derived growth factor (PDGF), tumor angiogenic factor (TAF), vascular endothelial growth factor (VEGF), corticotropin releasing factor (CRF), transforming There are growth factors α and β (TGF-α and TGF-β) interleukin-8 (IL-8), and granulocyte-macrophage colony stimulating factor (GM-CSF), interleukins, and interferons. It is not limited to.

Other drugs incorporated into the wound dressing of the present invention include acidic mucopolysaccharides, which include heparin, heparin sulfate, heparinoids, dermatan sulfate, pentosan polysulfate, cellulose, agarose. , Chitin, dextran, carrageenin, linoleic acid, and allantoin, but are not limited to these.

Anti-inflammatory and antithrombotic agents include endomedicin, heparin, indomethacin, ibuprofen, aspirin, choline salicylate, diflunisal, magnesium salicylate, magnesium choline salicylate, salsalate, flurbiprofen, fenoprofen, ketoprofen, naprosin, naproxen sodium, Oxaprozin, diclofenac sodium, diclofenac misoprostol, etodolac, indoside, ketorolac, nutmeton, sulindac, tolmetine, sulfinpyrazone, dipyridamole, ticlopidine, valdecoxib, rofecoxib, piroxicam, meloxicam, meclofenamic acid, mefenamic acid, mefenamic acid, mefenamic acid, mefenamic acid Cyclosporine micromarjon, chlorambucil, anagreri , Clopidogrel, and cilostazol. The antithrombotic agent may also be selected from the group consisting of heparin, ardeparin, enoxaparin, tinzaparin, danaparoid, elpyrden, and hirudin.

The therapeutically effective agent can be coupled to the biomaterial of the present invention, either physically or chemically, by methods known in the art.

Here, the word “comprising” means including whatever precedes “having”. Thus, the use of the term “comprising” indicates that the listed element is mandatory, but that other elements are optional and may or may not be present. ing. The word “consisting of” means that the wording “consisting of” includes anything before the wording of “consisting of” and is limited thereto. Thus, the word “consisting of” indicates that the described element is essential and that no other element can exist. In addition, the word “consisting essentially of” includes any element described as “to”, and other elements may interfere with the activities or behaviors specified in the disclosure of the element. It means that it is limited to elements that do not contribute or contribute. Thus, the word “consisting essentially of” indicates that the element described is essential, but other elements are optional and affect the activity or behavior of the element. It shows that the presence or absence of existence is determined by whether or not it exerts.

The invention is described in more detail below with reference only to the following non-limiting examples. However, it should be understood that the examples given below are for illustrative purposes only and should not be taken as limiting the generality of the invention described above. In particular, although the present invention describes in detail the treatment of the pericardium, aortic root, leaflets, and aortic wall from bovine, porcine, and marsupial sources, the findings of this application are based on these specific tissues Or it should be clearly understood that it is not limited to animal sources.
[Example 1]

Basic treatment of biomaterials

Adult black kangaroo hearts were collected in Western Autotralia by professional kangaroo hunters, packed in ice and transported to the laboratory within 4-6 weeks after death. The heart was washed twice with ice-cold 0.9% saline. The pericardium was removed from sticky fat and loose connective tissue and carefully washed. The aortic root, along with the aortic valve, was dissected from the heart and placed in 0.9% ice-cold saline containing 0.5 mM phenylmethylsulfonyl fluoride (PMSF). The pericardium is stored overnight in 4% C. ice cold 0.9% saline containing 0.5 mM PMSF, and the valved aortic root is 0.9 μm containing PMSF. Washed in% saline for 20 minutes.

A water-soluble alcohol-containing solution having an alcohol ethanol volume concentration of 60-80% v / v was prepared. The pericardium was immersed in this alcohol solution after storage at 4 ° C. overnight. The valved aortic root was left in an alcohol solution at about 5 ° C. for a minimum of 24 hours.

The pericardium and the valved aortic root were removed from the alcohol solution and flushed with 0.9% saline for about 10 minutes. During the washout period, the temperature of the washout liquid was maintained at about 10 ° C.

The pericardium and the valved aortic root were immersed in a solution of 0.625% glutaraldehyde containing 9.07 g / l of dihydrogen phosphate buffer in sterile deionized water. The pH of this glutaraldehyde solution was adjusted to 7.4 with sodium hydroxide. The pericardium and the valved aortic root were fixed in the glutaraldehyde solution at 1-5 ° C. for a minimum of 24 hours, and the proteins present in the collagen of the tissue were cross-linked.

The pericardium and the valved aortic root were removed from the glutaraldehyde solution and flushed with sterile 0.9% sodium chloride for about 15 minutes. During this washout period, the washout solution was maintained at about 10 ° C.

The pericardium and the valved aortic root were then treated by a procedure with two options. In the first procedure, the pericardium and the valved aortic root were immersed in a buffer-free solution containing 8 mg dicarboxylic acid per milliliter of deionized water. The pH of this solution was adjusted to pH 4.5 with the amount of dilute hydrochloric acid. The pericardium and the valved aortic root were immersed in this solution at about 45 ° C. for about 48 hours.

In the second procedure, the pericardium and the valved aortic root are a buffer-free solution containing a polyvalent cation such as magnesium, ferric salt or aluminum salt dissolved in deionized water, and pH 3 .5 soaked in the solution for about 60 minutes.

The biomaterial was then immersed in a 0.25% glutaraldehyde 0.25% solution containing 9.07 g / l potassium dihydrogen phosphate buffer in sterile deionized water or sterilized by any of the methods described below. . The pH of the aldehyde solution was adjusted to 7.4% with sodium hydroxide. This sterilization process was carried out at a temperature of about 45 ° C. for about 120 minutes.

As another sterilization method, the biomaterial was sterilized in an aqueous solution at 37 ° C. with 2% epoxypropane combined with 20% ethyl alcohol by weight for 24 hours. The sterilized tissue was then stored in buffered 0.2% glutaraldehyde plus 15% isopropanol.
[Example 2]

Effect of temperature on biomaterials

The modification temperature is an important measure of cross-linking stability, which reflects the strength, durability, and integrity of the material.

Bovine pericardium was obtained at a regional slaughterhouse in Western Australia and transported in ice. The pericardium was washed as described in Example 1.

The degree of cross-linking of bovine pericardium prepared according to the method described in Example 1 was compared to that of bovine pericardium (control pericardium) fixed with 0.625% buffered glutaraldehyde.

A pericardial sample strip (5 x 10 mm) representing each group is attached to an isotropic transducer (MLT0500 from AD Instruments, an Australian manufacturer) and interfaced to a PowerLab data acquisition system and a desktop computer. It was. The sample was always stretched with a load of 90 ± 5 g and immersed in an open water bath filled with 0.9% saline and temperature controlled. The temperature of this water tank was gradually raised from 25 ° C. to 95 ° C. at a rate of about 1.5 ° C./min. The contraction temperature was indicated by the abrupt deviation from the constant extension when the collagen material was denatured. A summary of the results (shrink temperature in degrees Celsius) is shown in Table 1.

［実施例３］

[Example 3]

Enzymatic degradation of biomaterials

Bovine pericardium prepared by the method described in Example 1 (treated pericardium) has a level of resistance to enzymatic degradation against 0.625% buffered glutaraldehyde (control) Compared to that of the pericardium).

As a pronase solution, a solution prepared by dissolving 100 mg of pronase (Streptomyces griseus) and 100 mg of calcium salt in 200 ml of HEPES buffer solution (0.01 M, pH 7.4) containing 0.1 M of glycine is prepared. It was. Fixed tissue samples were rinsed with deionized water for 3 minutes, wiped, dried at 70 ° C. overnight, and then weighed. These samples were incubated in the pronase solution at 50 ° C. for 24 hours. The remaining tissue sample was rinsed with deionized water, dried at 70 ° C. overnight, and then weighed. Pronase resistance to digestion was determined by the mass of the remaining tissue and expressed as a percentage of the tissue mass before digestion. A summary of the results is shown in Table 2.

［実施例４］

[Example 4]

Tensile strength of biomaterials

Tensile strength is an important measure of material strength and reflects the durability of the crosslinked structure.

The bovine pericardium (treated pericardium) prepared according to the method described in Example 1 has a tensile strength of bovine pericardium (control pericardium) fixed with 0.625% buffered glutaraldehyde. Compared to it.

The hydrostatic Zwick / Roell with a 10 k Newton load cell installed at a constant stretch rate of 50 mm / min is used for the tensile strength of the typical pericardial strip (8 × 80 mm) of both groups of tissues. ) Measured with a tensile tester (model 2010). The tensile strength and elongation at break were determined from the recorded load / elongation curve. A summary of the results is shown in Table 3.

［実施例５］

[Example 5]

Calcification characteristics of biomaterials

In order to evaluate the above-described process effectiveness in reducing mineralization of treated collagen-containing biomaterials, demonstration experiments of small and large animal models were conducted. In the first animal experiment, kangaroo leaflet tissue fixed only with 0.625% buffered glutaraldehyde and kangaroo aortic wall tissue (untreated tissue) were treated according to the method described in Example 1. Compared to leaflet tissue and kangaroo aortic wall tissue (treated tissue).

Both groups of aortic wall tissue samples (size 10 × 5 mm) and aortic leaflets were flushed with 0.9% saline for 5 minutes. The washed-out tissue was surgically implanted (one sample per rat) in a subcutaneous pocket formed in the center of the dorsal side wall of a growing (6 weeks old) male Wistar rat. After 60 days, the explanted tissue was dissected from surrounding host tissue and dried for 48 hours in a 90 ° C. biotherm incubator (Selby Scientific, Perth, Western Australia). The dry tissue was weighed and its calcium content was extracted with 5.0 ml of 6 nitrogen ultra-high purity hydrochloric acid (made by Mark, Perth, Western Australia) over 24 hours. Thereafter, the extractable calcium content was measured using an atomic absorption spectrophotometer (AA1275 manufactured by Varian), and calcium per 1 mg (dry weight) of the tissue was expressed in μg. A summary of these data is shown in Table 4.

The effectiveness of the new process for reducing calcification is readily apparent in Table 4. Treated tissue calcium levels were comparable to those normally present in non-fixed tissues, and therefore this new process is superior to standard glutaraldehyde immobilization alone. It is to illustrate that.
[Example 6]

Additional calcification experiments on sheep

In additional animal experiments, a kangaroo valved aortic conduit (treated tissue) fixed with 0.625% buffered glutaraldehyde as described in Example 1 was extracted as described in Example 1. Compared to valve aortic root (untreated tissue). The aortic root was flushed with 0.9% saline for 5 minutes and then surgically implanted into the pulmonary artery location of a young (4 months old) Merino Dorset hybrid sheep. These valved grafts were removed after 200 days and the calcium content of the leaflet and aortic wall tissues was measured with an atomic absorption spectrophotometer as described above.

A summary of the results (μg calcium per mg dry tissue) is shown in Table 5.

［実施例８］

[Example 8]

Further calcification experiments on swine-derived tissues

As a further animal experiment, an untreated porcine valved aortic conduit (untreated tissue) fixed with 0.625% buffered glutaraldehyde was treated according to the method described in Example 1 for porcine valved aorta. Compared to the conduit (treated tissue). The porcine valved aortic conduit was flushed with 0.9% saline for 5 minutes and then surgically implanted at the pulmonary artery location of a young (4 months old) Merino Dorset hybrid sheep. These valved grafts were removed after 200 days and the calcium content of the leaflet and aortic wall tissues was measured with an atomic absorption spectrophotometer as described above.

A summary of the results (μg calcium per mg dry tissue) is shown in Table 6.

［実施例８］

[Example 8]

Calcification of bovine pericardium

As a further animal experiment, the calcification potential of bovine pericardium fixed with 0.625% buffered glutaraldehyde (control pericardium) was treated with bovine pericardium treated according to the method described in Example 1 ( Compared to the calcification potential of the treated pericardium). A typical sample from each group was cut to a size of 1 × 1 cm and rinsed with 0.9% saline for 5 minutes. These samples were surgically implanted in a subcutaneous pocket formed in the center of the dorsal sidewall of adult (6 weeks old) Wistar rats. These tissues were removed after 60 days, the host tissue was removed, and the calcium content was then measured with an atomic absorption spectrophotometer as described above. A summary of the results (μg calcium per mg dry tissue) is shown in Table 7.

［実施例１０］

[Example 10]

Calcification of non-cellularized kangaroo pericardium

As another animal experiment, the calcification potential of kangaroo pericardium (control pericardium) fixed with 0.625% buffered glutaraldehyde was decellularized and treated according to the method described in Example 1. The calcification potential of the kangaroo pericardium (treated pericardium) was compared.

A representative sample from each group was cut to a size of 1 × 1 cm and rinsed with 0.9% saline for 5 minutes. These samples were surgically implanted in a subcutaneous pocket formed in the center of the dorsal sidewall of adult (6 weeks old) Wistar rats. These tissues were removed after 60 days, the host tissue was removed, and the calcium content was then measured with an atomic absorption spectrophotometer as described above. A summary of the results (μg calcium per mg dry tissue) is shown in Table 8.

［実施例１１］

[Example 11]

Recellularization of decellularized kangaroo pericardium

In a sixth animal experiment, the recellularization potential of kangaroo non-cellularized pericardium (control pericardium) fixed with 0.625% buffered glutaraldehyde was treated according to the method described in Example 1. Compared to the recellularization potential of kangaroo decellularized pericardium (treated pericardium). Kangaroo pericardium was decellularized in 0.25% Triton X100 and 0.25% sodium dodecyl sulfate for 24 hours at room temperature. Each group of pericardium test samples (n = 5) were cut to a size of 2 × 2 cm and rinsed with 0.9% saline for 5 minutes. These samples were seeded with 3.0 × 10 ⁵ / cm ² human fibroblasts under aseptic conditions. The seeded pericardium was cultured for 21 days at 37 ° C. in standard quiescent cell culture conditions. Cell growth was assessed microscopically every 7 days and classified according to the following visual criteria. 1) There are no growing fibroblasts on the surface of the matrix. 2) Less than 50% of the substrate surface is covered with fibroblasts (+). 3) Over 50% of the substrate surface is covered with fibroblasts (++). 4) The entire substrate surface is covered with multiple layers of fibroblasts (++++).

As a rapid colorimetric assay, the MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl tertrazolium bromide) test was performed on day 21 and grew on the control pericardium The absence of fibroblasts and the presence of growing fibroblasts in the treated pericardium were confirmed (for example, see Zund et al., Eur J Cardiothorac Surg. 1999, 15 (4), Pp. 519-524). A summary of the results is shown in Table 9.

［実施例１２］

[Example 12]

Cross-linking stability of kangaroo tissue

Two types of kangaroo tissue, aortic wall tissue and kangaroo pericardium were treated according to the procedure outlined in Example 1 up to step (b), including step (b), ie, alcohol solution pretreatment Later, glutaraldehyde crosslinking was performed. These treated tissues were compared to tissues fixed with glutaraldehyde, ie tissues that had not been pretreated with alcohol as well as “fresh” tissues. 1 and 2 show pericardial crosslinking stability (FIG. 1) and aortic wall tissue crosslinking stability (FIG. 2). From the figure, it can be seen that ethanol pre-treatment has a significant effect on cross-linking stability, ˜85 ° C. to 86 ° C. These data can also be seen in Table 10.

［実施例１３］

[Example 13]

Cross-linking stability and mineralization behavior in a subcutaneous rat model.

This experiment shows the cross-linking stability and mineralization behavior of porcine tissues (cusps and walls) treated with the method disclosed in Example 1, control tissue fixed with glutaraldehyde, and commercial freestyle. A (Freestyle®) biological prosthetic tissue was compared to a commercially available PrimaPlus® biological prosthetic tissue.

Fresh porcine valved aortic roots were collected and carried in 4 ° C. phosphate buffered saline (PBS, 0.1 M, pH 7.4). Representative aortic valve cusps (n = 30) and aortic wall samples (n = 30, 10 mm × 15 mm) were removed from the aortic root and divided into two groups. Group I included representative valve cusps (n = 15) and representative aortic wall samples (n = 15, 10 mm × 15 mm) and were stored in 0.25% buffered glutaraldehyde. Group II includes a representative valve cusp (n = 15) and a representative aortic wall sample (n = 15, 10 mm × 15 mm), exposed by the method disclosed in Example 1, and 0.25% buffered glutar Stored in aldehyde. For comparison, Group III was divided into a freestyle (Freestyle®) bioprosthetic tissue valve cusp (n = 15) and aortic wall sample (n = 15, 10 mm × 15 mm) and PrimaPlus (registered). (Trademark)) consisting of a valve cusp (n = 15) of biological prosthetic tissue and an aortic wall sample (n = 15, 10 mm × 15 mm).

A shrinkage temperature measurement method was used to evaluate collagen cross-linking stability of tissues (Levi et al., Am. J. Pathol., 1986, 122, 71-82). Each group of cusps and aortic wall sample tissue strips (5 × 10 mm, n = 10) are attached to an isotropic transducer (MLT0500, AD Instruments, Australia), PowerLab data acquisition system and desktop PC Was interfaced to.

The sample was kept stretched at a load of 90 ± 5 g, immersed in an open water bath filled with 0.9% saline and temperature controlled. The water bath temperature was gradually raised from 25 ° C. to 95 ° C. at a rate of about 1.5 ° C./min. The contraction temperature was indicated by the abrupt deviation from the constant extension when the collagen material was denatured.

（１グループに付きｎ＝１０）
値は平均値±標準誤差
^＊ｐ＜０．０５（試験対対照、フリースタイル、プリマプラス） The cusp and aortic wall contraction temperatures are listed in Table 1. There was no significant difference between the control, the test procedure of Example 1 (“Test”), Freestyle (Freestyle®), and Primaplus (PrimaPlus®) cusps. The aortic wall tissue treated with the test procedure is significantly higher (p <0. 0) compared to the control, freestyle (Freestyle®), and PrimaPlus (PrimaPlus®) respective aortic wall tissue. 05) Indicates shrinkage temperature.
Contraction temperature of valve cusp and aortic wall tissue (℃)

(N = 10 per group)
Values are mean ± standard error
^* P <0.05 (Test vs. Control, Freestyle, PrimaPlus)

The resistance to digestion with protein enzyme is based on Girardot and Girardot's method (J. Heart valve Dis., 1996, 122: 71-82). Pronase solution in which 10 mg of Pronase E (Streptomyces griseus type XIV, Sigma) and 100 mg of calcium chloride are dissolved in 200 ml of HEPES buffer solution (0.01 M, pH 7.4) containing 0.1 M of glycine Was prepared. Fixed tissue samples were rinsed with deionized water for 3 minutes, wiped, dried at 70 ° C. overnight, and then weighed. These samples were incubated in a pronase solution at 50 ° C. for 24 hours. The remaining tissue sample was rinsed with deionized water, dried at 70 ° C. overnight, and then weighed. The resistance to digestion against pronase was determined by the mass of the remaining tissue and expressed as a percentage of the tissue mass before digestion.

The resistance against enzymatic degradation is shown in FIG. The cusp tissues of the test procedure, Freestyle (Freestyle®), and PrimaPlus (PrimaPlus®) each showed a significant (p <0.0001) resistance to proteolysis compared to the control. ) Increased (FIG. 3A). There is no significant difference between the cusp tissues of the test procedure, Freestyle (Freestyle®), and PrimaPlus (PrimaPlus®).

Aortic wall tissue treated with the test procedure shows resistance to proteolysis (p = NS) comparable to the control tissue, freestyle (Freestyle®) and Primaplus (PrimaPlus®), respectively. The resistance was significantly higher (p <0.01) than the wall structure (Fig. 3B).

Young male Wistar rats (weight 150-200 g) were divided into two groups. The first group (n = 10) was transplanted with cusps and the second group (n = 10) was transplanted with aortic walls. For each animal, one sample from each of the four groups of tissues was transplanted, for a total of 80 transplants.

Rats were anesthetized with pentobarbital (Nembutal®, 45 mg / kg, ip). The dorsal muscle region was shaved and disinfected with 15% dilute chlorhexidine gluconate (made by ICI Pharmaceutical, Perth, Western Australia) and ethanol (made by Merck Chemical, Perth, Western Australia).

The implant was thoroughly rinsed with deionized water for 2 minutes to remove the sticky material and then implanted through a 2.5 cm incision into a subcutaneous bag on the muscle wall of the back. The incision was closed with 5-0 prolane suture.

Rats died after 8 weeks of overdose of barbituric acid (Euthenase®), and the dorsal muscle wall, including the subcutaneous graft, was removed for quantitative and qualitative tissue calcium analysis. I was allowed. Each collected sample was divided into anatomically symmetric half. One half was subjected to atomic absorption spectrometry and the other half was fixed with 10% buffered formaldehyde and processed for histology.

Fixed samples were embedded in paraffin wax and separated at 3 μm intervals, and then quantitative calcium analysis was performed by the von Kossa calcium detection method. Histological examination was performed using an Olympus BHS optical light microscope.

All groups of explant samples were excised and isolated from surrounding host tissue and dried for 48 hours in a 90 ° C. biotherm incubator (Selby Scientific, Perth, Western Australia). The dried sample was light and its calcium content was extracted for 24 hours with 5.0 ml of 6N ultra-high purity hydrochloric acid (Mark, Perth, Western Australia) at 75 ° C. The extractable calcium content was measured using an atomic absorption spectrophotometer (AA1275 manufactured by Varian) and expressed in μg of calcium per 1 mg (dry weight) of tissue.

Histological examination showed the presence of intense endogenous calcification in the explanted control samples (data not shown). No visible calcification was observed on ADAPT, freestyle or Primaplus valve cusps (data not shown).

The explanted aortic wall tissue had some calcified sites in the media in the control sample (data not shown). There was no visible calcification in the explanted test aortic wall tissue (data not shown). Mild calcification of the media was observed in explanted freestyle aortic wall tissue and explanted Primaplus aortic wall tissue (data not shown).

The quantitative tissue calcium level of explanted cusps is shown in FIG. 4A. A control sample fixed only with glutaraldehyde showed the highest level of calcium in this model (92.37 ± 7.9 μg / mg). The calcium level of the tissue treated by the method of Example 1 was 2.09 ± 0.22 μg / mg per mg of tissue, whereas Freestyle (Freestyle®) was 2.03 ± 0.29 μg / mg. mg, Primaplus (PrimaPlus®) was 1.54 ± 0.17 μg / mg. This means that the treated tissue has significantly reduced calcium levels (p <0.001) compared to the control sample. There is no significant difference between the tissue calcium levels of Example 1, Freestyle (Freestyle®) and PrimaPlus (PrimaPlus®).

The quantitative tissue calcium level of the explanted aortic wall sample is shown in FIG. 4B. The calcium content of the aortic wall sample treated by the method of Example 1 (4.86 ± 0.12 μg / mg per mg tissue) is that of the control sample (120.11 ± 7.48 μg / mg tissue) Significantly lower (p <0.001) (95.9% lower). Freestyle (Freestyle®) and PrimaPlus (PrimaPlus®) aortic wall samples showed a 47.8% and 51.95% reduction in calcification, respectively.

These data suggest that the method disclosed in Example 1 is effective in reducing both porcine caps and wall tissue calcification in the subcutaneous rat model. These data also suggest that accelerated cross-linking plays an important role in minimizing aortic wall calcification.

A diagram showing kangaroo pericardial tissue, showing glutaraldehyde immobilized tissue pretreated with alcohol compared to glutaraldehyde immobilized tissue without pretreatment with alcohol and "fresh" tissue. A diagram showing kangaroo aortic tissue showing glutaraldehyde-immobilized tissue pretreated with alcohol compared to "fresh" tissue with glutaraldehyde-immobilized tissue without pretreatment with alcohol. Figure (A) shows the enzymatic degradation resistance of porcine leaflet tissue, and Figure (B) shows that of porcine aortic wall tissue. Quantitative calcium levels of explanted porcine leaflet tissue 8 weeks after subcutaneous in rat model are shown in (A) and porcine aortic wall tissue in (B).

Claims (45)

A method of making an implantable anti-calcified biomaterial comprising:
(A) exposing the collagen-containing material to an alcohol-containing solution for at least 24 hours;
(B) exposing the raw material treated by step (a) to a cross-linking agent;
(C) the raw material treated in step (b), both of the fixed and non-fixed crosslinker components present in the raw material after step (b), or one of them, Exposing to an acidic solution containing an aminocarboxylic acid that can be inactivated and / or modified, and / or
And steps (b) and (c) are sequentially performed after step (a). The method of claim 1, further comprising the step of flushing between steps (a) and (b) and / or steps (b) and (c). The method according to claim 1 or 2, further comprising a step of flushing after step (c). The method according to claim 2 or 3, wherein the step of washing off the raw material is carried out using 0.9% physiological saline without phosphorus. The method according to any one of claims 1 to 4, wherein the collagen-containing raw material is extracted from an animal. 6. The method of claim 5, wherein the animal is selected from the group consisting of sheep, cows, goats, horses, pigs and humans. The method according to any one of claims 1 to 6, wherein the raw material is a cultured tissue and is a prosthesis containing an extracellular matrix obtained from an animal or a regenerated tissue. The raw material is cardiovascular tissue, pelvic floor tissue, heart tissue, heart valve, aortic root, aortic wall, aortic apex, pericardial tissue, connective tissue, soft or solid organ matrix, skin tissue, vascular tissue, dura mater The method according to any one of claims 1 to 11, which is a cell tissue selected from the group consisting of cartilage, pericardium, ligament, tendon, blood vessel, umbilical tissue, bone tissue, fascia, and submucosa or skin. . The method according to any one of claims 1 to 8, wherein the alcohol-containing solution used in step (a) has one or more water-soluble alcohols. The method according to claim 9, wherein the water-soluble alcohol is a C ₁ -C ₆ lower alcohol. Those wherein C ₁ -C ₆ lower alcohol, methanol, ethanol, cyclohexanol, isopropanol, propanol, butanol, pentanol, isobutanol, secondary butanol, selected from tertiary butanol and the group consisting of The method of claim 10, wherein The method according to any one of claims 1 to 11, wherein the alcohol-containing solution has less than 100% alcohol in an unbuffered aqueous solvent. 13. A method according to any preceding claim, wherein step (a) is performed for at least 36 hours. 14. A method according to any preceding claim, wherein step (a) is carried out for at least 48 hours. 15. The cross-linking agent is selected from the group consisting of carbodiimide, polyepoxy ether, divinyl sulfone (DVS), genipin, polyaldehyde, diphenylphosphoryl azide (DPPA), and combinations thereof. The method in any one of. The method according to claim 15, wherein the polyaldehyde is glutaraldehyde. The method according to any one of claims 1 to 16, further comprising a sterilization step after step (c). The method according to any one of claims 1 to 17, wherein the step (a) is performed at a temperature of about 5 ° C. The method according to any one of claims 1 to 18, wherein the step (c) is performed at a temperature between 5 ° C and 55 ° C. The method according to claim 1, wherein the acidic solution is capable of cross-linking activated carboxyl groups and amine groups on collagen to form an amide bond. The method according to any one of claims 1 to 20, wherein the aminocarboxylic acid is selected from the group consisting of L-histidine, L-arginine, L-lysine, L-glutamic acid and L-aspartic acid. The method according to any one of claims 1 to 21, wherein the alcohol-containing solution, the cross-linking agent, and the acidic solution are all free of a buffer. 23. An implantable anticalcified biomaterial produced by the method of any of claims 1-22. 24. The implantable anti-calcified biomaterial of claim 23, wherein the biomaterial contains less than about 50 μg calcium per mg biomaterial, and further increases the calcium content per mg biomaterial above 50 μg. Biomaterial that can be transplanted for at least 200 days without letting go. 25. The implantable anti-calcified biomaterial of claim 24, wherein the biomaterial prior to transplantation has less than about 20 [mu] g calcium per mg biomaterial. 25. The implantable anti-calcified biomaterial of claim 24, wherein the biomaterial prior to transplantation has less than about 10 [mu] g calcium per mg biomaterial. 27. An implantable biological device comprising the implantable anticalcified biomaterial according to any of claims 23 to 26. 28. The biological device of claim 27, wherein the biomaterial is coated on a surface of the device. 29. A biological device according to claim 27 or 28, further comprising at least a second coating. 30. The biological device of claim 29, wherein the at least second coating comprises one or more of an antibacterial agent, an antiviral agent, an antifungal agent, a growth factor, an antidehydrating agent, or a disinfectant. The antibacterial agents are isoniazid, ethambutol, pyrazinamide, streptomycin, clofazimine, rifabutin, fluoroquinolones, ofloxacin, spalfloxacin, rifampicin, azithromycin, clarithromycin, daphorn, tetracycline, erythromycin, ciprofloxacin, doxycycline, doxycycline, ampicillin , Amphotericin B, ketoconazole, fluconazole, pyrimethamine, sulfadiazine, clindamycin, lincomycin, pentamidine, atovaquone, paromomycin, diclazaryl, acyclovir, trifluorouridine, foscarnet, penicillin, gentamicin, ganciclovir, iatroconazole, miconazole, zinc pyrithizine , Gold, platinum, silver, zinc and copper Selected from the group consisting of, but not limited to, heavy metals, and salts of chlorides, bromides, iodides, periodates, and the like, including the supported complexes, and other forms 32. The biological device according to claim 30, wherein: Said growth factor drug is hydroxyapatite, basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), nerve growth factor (NGF), epidermal growth factor (EGF), insulin-like growth factor 1 and 2 (IGF-1 and IGF-2), platelet derived growth factor (PDGF), tumor angiogenic factor (TAF), vascular endothelial growth factor (VEGF), corticotropin releasing factor (CRF), transforming growth factors α and β ( 30. Selected from the group consisting of TGF-α and TGF-β) interleukin-8 (IL-8), and granulocyte macrophage colony stimulating factor (GM-CSF), interleukins, and interferons The biological device as described. The at least second coating further comprises polylactic acid, polyglycolic acid, polylactic acid-polyglycolic acid copolymer, polydioxanone, polycaprolactone, polypeptides, polycarbonates, polyhydroxybutyrate, poly (alkylene sulfonate). Acid salt), copolymers of vinyl acetates with unsaturated carboxylic acids, water-soluble or water-dispersible cellulose derivatives, ethylene oxide polymers, polyacrylamide, collagen, gelatin, poly (orthoesters), amino acid polyamides, 30. The biodevice of claim 29, comprising a bioabsorbable material selected from the group consisting of polyvinyl alcohol, polyvinyl pyrrolidone, polyetheretherketone, tricalcium phosphate, and mixtures thereof. The biological device is an artificial heart, built-in or external cardiac assist device, heart valve prosthesis, annuloplasty ring, skin graft, vascular graft, vascular stent, structural stent, vascular short circuit, cardiovascular short circuit, dura mater transplant Fragment, cartilage graft, cartilage graft, pericardial graft, ligament prosthesis, tendon prosthesis, bladder prosthesis, cotton ball, suture, permanent indwelling percutaneous device, surgical patch, cardiovascular stent, coated stent, and coating The biological device according to any one of claims 27 to 33, wherein the biological device is selected from the group consisting of catheters. 35. The biological device of claim 34, wherein the biological device is a heart valve prosthesis. 36. The biological device according to any one of claims 27 to 35, wherein the biological device further has a tissue fragment. The biological device according to claim 36, wherein the tissue fragment is collected from an animal. 38. The biological device according to claim 37, wherein the animal is selected from the group consisting of human, cow, pig, dog, deer and kangaroo. 39. A biological device according to any of claims 27 to 38, wherein the biological device further comprises a synthetic analog of tissue. 27. A biocompatible implant having a biocompatible scaffold with an implantable anti-calcified biomaterial according to any of claims 23 to 26. A tissue injury repair kit,
(A) a sterilization container containing one or more biological devices according to any one of claims 27 to 39;
(B) a description of the injury subject;
A tissue injury repair kit comprising: The kit further comprises:
(C) a collection tool for collecting at least one viable tissue sample from the injured subject;
42. The kit of claim 41, comprising: 43. The kit of claim 42, further comprising at least one reagent for maintaining the viability of the at least one tissue sample. 43. The kit of claim 42, wherein the collection tool further comprises a processing tool for splitting at least one tissue fragment from the tissue sample in a sterile condition. 27. A wound dressing comprising the implantable anti-calcified biomaterial according to any of claims 23 to 26.

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